1a. Objectives (from AD-416):
Objective 1: Discover and analyze the function of genes involved in pathogenicity of foliar fungal pathogens of wheat and other grain crops. Sub-objective 1a. Provide gene-expression annotations for the approximately 44% of the genes in the M. graminicola genome that have no predicted function, particularly those that share conserved domains of unknown function in other fungi. Sub-objective 1b. Analyze genes expressed under many conditions including during early, transitional and late stages of pathogenicity to identify those that may be important for disease development and other biological processes. Sub-objective 1c. Test the function of candidate pathogenicity genes or others involved in important biological processes by knocking out their expression and analyzing changes in phenotype. Objective 2: Characterize the genetic and biochemical bases for resistance of wheat to the foliar fungal pathogens responsible for Septoria and Stagonospora leaf blotches. Sub-objective 2a. Analyze genes expressed at several time points after inoculation with the pathogens to identify those associated with the major-gene resistance response of wheat. Sub-objective 2b. Analyze genes expressed at several time points after inoculation with the pathogens to identify those associated with non-host resistance responses of wheat and barley. Sub-objective 2c. Test the function of candidate resistance-associated genes by virus-induced gene silencing (VIGS) or RNA interference (RNAi). Objective 3: Develop genetic markers to be used by associated breeding programs in the development of disease-resistant germplasm of wheat and other grain crops. Sub-objective 3a. Develop large recombinant-inbred populations segregating for the Stb2 and Stb3 genes for resistance to Septoria tritici blotch in wheat and identify additional closely linked molecular markers. Sub-objective 3b. Develop mapping populations to identify quantitative resistance against the Septoria pathogens of wheat. Sub-objective 3c. Identify and validate molecular markers linked to quantitative resistance against the Septoria pathogens of wheat. Sub-objective 3d. Test Chinese wheat cultivars for resistance against the Septoria pathogens of wheat.
1b. Approach (from AD-416):
The preferred approach will be sequencing of messenger RNAs produced during several stages of pathogenesis by Mycosphaerella graminicola and Stagonospora nodorum on wheat, and of non-host resistance responses on barley. Numerous libraries will be analyzed from as many treatments as possible to obtain the greatest number of expressed genes, which was shown to be very effective in a previous analysis of EST sequences. Some treatments will consist of the pathogen exclusively in pure culture to identify genes involved in fungal development and responses to light, while others will include the pathogen in its host to identify genes involved in fungal pathogenicity and in host resistance. Most experiments will be done with isolate IPO323 of M. gramincola, the strain that was sequenced. Availability of a completely sequenced genome for M. graminicola provides a unique opportunity to analyze global gene expression to identify candidate genes for pathogenicity, sporulation, mating, light reception/regulation and other important biological processes. For some host-pathogen interactions isolates T48 from Indiana and isolate Pasco from Australia may be used. Isolate T48 is avirulent on all resistance sources tested so far but gives good infection on susceptible controls. It was used to map genes Stb1, Stb4 and Stb8. The Pasco isolate was used to map resistance genes Stb2 and Stb3 and can be used as a backup in case of problems with one of the other isolates.
3. Progress Report:
Objective 1. Genetic variability of dispensable chromosomes was analyzed within populations of the wheat pathogen Mycosphaerella graminicola. This fungus has at least eight chromosomes that can be lost with no obvious effects on fitness so apparently are dispensable, yet appear to have been retained within populations for more than 10,000 years so must be beneficial under at least come conditions. We previously designed six genetic markers that span all eight dispensable chromosomes. Analysis of these markers on isolates of the pathogen from around the globe revealed that individuals could be missing all or most of many dispensable chromosomes but that the content of each chromosome was conserved within each population. We expected to find variation for presence of particular dispensable chromosomes among populations so this result was unexpected. This most likely means that genes on the dispensable chromosomes have a positive effect on fitness that is not constant, so they can be lost in particular individuals when the selective benefit is not needed, but are retained within the population due to periods of beneficial selection. An experimental-evolution experiment was completed to test whether fungicides could affect the asexual transmission of dispensable chromosomes in M. graminicola. Dispensable chromosomes were lost during all treatments, including the control, but the rate of loss was increased by fungicide treatments. This indicates that treatment with fungicides may increase the rate of change of dispensable chromosomes, possibly allowing the fungus to adapt with higher resistance. Objective 2. Work on making isogenic lines for the Stb2 and Stb3 genes for resistance to M. graminicola is continuing. We now have both resistance genes in a common susceptible background of wheat. We hope to complete one more round of purification before those lines can be used experimentally. Objective 3. Crosses were made between lines containing the Stb2 and Stb3 genes for resistance to M. graminicola and the resulting F1 plants were inoculated with Indiana and Australian isolates of the pathogen. The Australian isolate was included because it was used to genetically map the Stb2 and Stb3 resistance genes previously. The F1 plants containing the Stb2 gene were susceptible but those with Stb3 were resistant, indicating that the two genes were recessive and dominant, respectively. This is particularly interesting because it indicates that the genes mostly likely function by different mechanisms. The Stb2 genetic locus actually may be a dominant gene for toxin sensitivity, making the resistance appear recessive.
1. Analysis of dispensable chromosomes within populations of Mycosphaerella graminicola. The genome sequence of the fungus Mycosphaerella graminicola, which causes Septoria tritici blotch of wheat, contained eight chromosomes that can be lost with no apparent effect on fitness of the organism so are dispensable, yet nothing is known about their presence within field populations or their function. To address this problem, ARS researchers at West Lafayette, Indiana, used the genome sequence of M. graminicola to design six molecular markers that spanned each dispensable chromosome, for 48 markers across all eight dispensable chromosomes. Presence or absence of all 48 markers were determined for populations of the fungus in North America and, in collaborative research with a scientist in the Netherlands, with additional populations throughout the world. Fifty-three progeny isolates from a sexual cross and 20 lines derived by 10 generations of asexual transfers also were analyzed to test the stability of dispensable chromosomes during sexual and asexual reproduction. The results showed that dispensable chromosomes are highly variable and unstable. The content of each dispensable chromosome was conserved within field populations, but not within individuals, some of which had lost almost every dispensable chromosome. Almost 90% of the sexual progeny had molecular marker patterns that were different from the parents and could not be explained by simple inheritance of dispensable chromosomes. Forty percent of the asexual progeny lines had lost one or more dispensable chromosomes, indicating that they are unstable during asexual reproduction. This information will be of interest to plant pathologists trying to control septoria tritici blotch, to fungal geneticists trying to understand the molecular basis for host-pathogen interactions, and to evolutionary biologists trying to understand fungal genome evolution.
Hane, J.K., Rouxel, T., Howlett, B., Kema, G.J., Goodwin, S.B., Oliver, R.P. 2011. Mesosynteny; A novel mode of chromosomal evolution peculiar to filamentous Ascomycete fungi. Genome Biology. 12:R45.